Simple lateral flow immunoassay devices have been developed and commercialised for detection of analytes in fluid samples, see for example EP291194. Such devices typically comprise a porous carrier comprising a dried mobilisable labelled binding reagent capable of binding to the analyte in question, and an immobilised binding reagent also capable of binding to the analyte provided at a detection zone downstream from the labelled binding reagent. Detection of the immobilised labelled binding at the detection zone provides an indication of the presence of analyte in the sample.
Alternatively, when the analyte of interest is a hapten, the immunoassay device may employ a competition reaction wherein a labelled analyte or analyte analogue competes with analyte present in the sample for an immobilised binding reagent at a detection zone. Alternatively the assay device may employ an inhibition reaction whereby an immobilised analyte or analyte analogue is provided at a detection zone, the assay device comprising a mobilisable labelled binding reagent for the analyte.
A sandwich immunoassay is often the assay of choice when detecting analytes. However, a sandwich assay is not always possible, for example in the case of small molecules such as haptens which may not be large enough to allow the simultaneous binding thereto of two different binding partners. A dose-response curve prepared using a typical lateral flow device employing a sandwich immunoassay shows increasing levels of signal with increasing analyte up to the point where at higher analyte levels the curve tends to plateau. At yet higher analyte levels, the signal begins to decrease due to preferential capture at the detection zone of analyte which has not yet bound to labelled reagent. This phenomenon is known as the hook effect. Thus sandwich immunoassays exhibit a limited assay range due to the fact that the signal amount or intensity observed at higher analyte levels may be the same, or even less, than that observed at lower analyte levels.
A competition or inhibition assay typically provides a high signal at zero or low levels of analyte. At increasing levels of analyte the signal level may still be high depending upon the amount of labelled binding species present compared to the amount of analyte. At still increasing levels of analyte, the signal starts to decrease as unbound analyte either competes with labelled analyte or analyte analogue for the immobilised binding reagent or binds to labelled binding reagent, lowering binding of the labelled binding reagent at the detection zone.
So, use of sandwich assays to measure analyte over an extended range may provide issues with respect to the hook effect. High analyte concentrations start producing a reduction in assay signal. Competition or inhibition assays result in the depletion in assay signal at high analyte concentrations and thus offer a limited range over which analyte can be measured.
Thus the above assay methods are not suitable for measuring levels of analyte over an extended analyte range.
US2005/0112780 discloses an assay device and method for extending the dynamic detection range of assay devices comprising a flow through porous carrier comprising a detection zone and a compensation zone provided downstream from the detection zone. The detection involves a first binding reagent which binds a detection probe to generate a detection signal having an intensity proportional to the amount of analyte, and the compensation zone comprises a second capture reagent which binds a detection probe to generate a signal which is inversely proportional to the intensity of the detection signal. The assay may further comprise a third calibration zone which generates a signal. The first binding reagent may be selected from a group including an antigen, hapten or streptavidin. The first and second binding reagents may be chosen from a number of species including an antigen, hapten or streptavidin.
US2004/0197820 discloses a flow through porous carrier assay device for reducing the hook effect comprising a detection zone wherein the device may include a downstream calibration zone.
US2006/0019404 discloses an assay device with an extended dynamic range comprising a lateral flow test-strip comprising a plurality of detection zones with a progressively decreased sensitivity to analyte concentration. The assay device may comprise two carriers each having a plurality of detection zones. The amount of label/signal present at the plurality of detection zones is detected to determine the analyte concentration.
EP462376 discloses an assay device comprising a capture site and a conjugate recovery site wherein the conjugate recovery site receives and binds said conjugate or conjugate complexes which migrate through said capture site and wherein immobilised conjugate at both the conjugate recovery site and capture site is detected to determine the amount of the analyte of interest.
The present inventors have shown that for assay devices wherein multiple detection zones for the detection of an analyte are provided on the same porous carrier, the binding at an upstream detection zone may change the binding characteristics at a downstream detection zone and that any variation in binding at an upstream detection zone may cause a compounded variation of binding at a downstream detection zone. This is especially so at higher analyte concentration levels and can give rise to poor assay precision. Furthermore, it has been found that cross-binding may occur between the respective binding reagents present in the detection zones during running of the test and cross-binding has also been observed during manufacture of the devices and whilst they are stored in the dry state. This was shown to have an impact on the levels of assay precision and sensitivity. These problems do not appear to have been recognised previously in the prior art.
It is an object to provide an improved assay device, kit and method for extending the analyte range of an assay.